Neuronal apoptosis is an important aspect of nervous system development and a component of neuronal injury and disease. The most generally accepted model of the developmental regulation of neuronal death states that limiting quantities of target-derived neurotrophic support control the optimum number of neuron-target interactions (1). Neurotrophins also play a role in ameliorating the effects of oxidative stress and many forms of neuronal injury (2,3).
In an effort to define the mechanisms of neurotrophin action in neuronal survival, two model systems, the PC12 pheochromocytoma cell line and cultured primary sympathetic neurons, have been exploited. The PC12 cell line was initially derived from a rat adrenal medullary pheochromocytoma (4). When grown in serum-containing medium, PC12 cells divide and resemble precursors of adrenal chromaffin cells and sympathetic neurons. Upon addition of NGF, these "naive" cells, gradually attain the phenotypic properties of sympathetic neurons. Both naive and neuronally-differentiated PC12 cells undergo apoptosis upon removal of trophic support (i.e. serum or serum/NGF)(5,6). The response of PC12 cells to withdrawal of trophic support is quite analogous to that of sympathetic neurons. In vivo (7-9) and in vitro (10,11) evidence demonstrate that sympathetic neurons require NGF for survival. Studies (6,12,13,14) have shown that both PC12 cells and sympathetic neurons undergo apoptotic death upon NGF deprivation.
Although the mechanisms by which neurotrophins suppress apoptosis are not fully understood, it has been hypothesized that neurotrophins prevent apoptotic death by acting to coordinate cell cycle progression and/or prevent inappropriate cell cycle reentry (12,15-17). Accordingly, this hypothesis predicts that cells which attempt to enter or traverse the cell cycle without a set of proper mitogenic signals will undergo apoptosis. In support of this model, numerous observations of apoptosis in the presence of conflicting cell cycle signals have been reported in non-neuronal systems (18-21). The cell cycle/apoptosis hypothesis was applied to interpret the characteristics of apoptotic death in PC12 cells, sympathetic neurons, and other cells of neuronal origin (15). In this view, withdrawal of serum from naive, proliferating PC12 cells leads to an uncoordinated and disastrous attempt to continue to cycle, whereas in post-mitotic differentiated PC12 cells and sympathetic neurons, withdrawal of NGF results in an inappropriate attempt to reenter the cell cycle and consequent death.
Previous findings have provided some evidence for this interpretation and for the cell cycle/apoptosis model in neuronal cells. While apoptotic death of sympathetic neurons and post-mitotic PC12 cells is delayed by protein synthesis inhibitors (11,14,23), such inhibitors do not block cell death of naive PC12 cells (24). One interpretation of this discrepancy is that the proteins needed for apoptosis are regulators of the general cell cycle mechanism. Since naive PC12 cells continually synthesize cell cycle proteins, they may utilize a preexisting pool of cell cycle regulators to enter the cell cycle even in the absence of new protein synthesis. Without appropriate coordinating mitogenic signals such as provided by growth factors, apoptosis would result. In contrast, post-mitotic cells would require de novo synthesis of cell cycle proteins prior to inappropriate cell cycle reentry. In accordance with this view, Freeman et al. (17) showed that NGF removal from sympathetic neurons results in an induction of the cell cycle regulatory protein cyclin D1 along with transcription factors, c-fos and c-jun (25), typically induced prior to cell division. Furthermore, the activation of another cell cycle protein, cdc2, has been reported in differentiated PC12 cells as a consequence of NGF withdrawal (26). It has also been reported that expression of SV40 T antigen in Purkinje cells results in apoptotic death (27) concurrent with DNA synthesis.
Initial attempts to test the cell cycle/apoptosis model by blocking cell cycle progression have produced additional support for this hypothesis. Induction of dominant-negative ras expression in both naive and post-mitotic PC12 cells inhibits cell cycle progression and death induced by withdrawal of trophic support (15). A similar correlation between survival and blockade of cell cycle has been shown in PC12 cells and sympathetic neurons treated with N-acetyl cysteine (NAC) (28). In these cases, the mechanisms by which the cell cycle is inhibited are unknown.
The cyclin dependent kinase (cdk) family, which among others includes cdk2-4/6, and cdc2 (cdk1), is an important group of cell cycle regulatory molecules whose inhibition represents a more defined means to block cell cycle progression or reentry. cdc2 is a well characterized M-phase regulator and may also serve to mediate progression through the S-phase (30). cdk2 and 3 activities are required for progression through the G1/S phases of the cycle (30,31). The present invention discloses the effects of inhibitors of the cdk family of kinases on PC12 cells and sympathetic neurons after withdrawal of trophic factor.